Delayed differentiation of HL-60 cells following exposure to hypoxia

Hemorrhagic shock and hypoxia have been shown to alter immune and hematopoietic functions. Cellular hypoxia is thought to be the primary defect and has been shown to induce a variety of biological alterations. In this study, we examined if this defect is at the stage of terminal differentiation with the myelomonocytic cell line HL-60.

After hypoxia, HL-60 cells were induced with 1.25% dimethyl sulfoxide (DMSO) to differentiate toward neutrophils (PMN). The ability to differentiate was evaluated by nitroblue tetrazolium staining. The function of the differentiated cells was determined by intracellular calcium levels after exposure to different chemotactic factors, and levels of Id-2 mRNA, a factor associated with terminal differentiation of myeloid cells, were assessed with Northern analysis.

At 48 h following exposure to hypoxia, HL-60 differentiation was significantly blunted (hypoxia 51 +/- 1%, normoxia 69 +/- 1%; P < 0.001). Intracellular calcium levels in DMSO-treated cells stimulated with 1 microM bacterial tripeptide, fMLP, were significantly reduced in the hypoxic cells (381 +/- 11 nM vs 449 +/- 10 nM; P < 0.01). No difference was noted for two other chemotactic factors, C5a and platelet-activating factor. Using Northern analysis to determine the levels of Id-2 mRNA, we demonstrated that hypoxia reduced the levels by 20% over normoxic cells.

This study demonstrates that hypoxia blunts the differentiation of HL-60 cells to PMN. This altered function of hypoxia appears to be reversible since hypoxia prolonged the time for HL-60 cells to differentiate and this may be partly explained by the premature downregulation of Id-2 expression.